Scalable biomimetic SARS-CoV‑2 nanovaccines with robust protective immune responses

coronavirus 2 (SARS-CoV-2) infection requires rapid development of vaccines matching the pace of virus mutation. While the ﬁ rst-generation of nucleic acid vaccines have been successful, subunit vaccines carry far fewer safety concerns and also have shown promise in clinical trials. 1 Innovations in biomaterials science and nanotechnology have produced nanoparticulate subunit vaccines that can target the Receptor-Binding Domain (RBD) of the spike protein to generate a robust immune response for immuniza- tion. 2,3 However, the time-consuming and costly recombinant SARS-CoV-2 RBD production, together with challenging quality control, diminish the appeal of adopting this approach for developing vaccine candidates for mass immunization. A vaccine design that is more economical, versatile, and manufacturable would be needed to combat the evolving COVID-19 pandemic. Enthused with the broad antibody generation and robust T cell response generated by inactivated or live-attenuated virus, we propose a vaccine design comprising a cell membrane-coated nanoparticle, where the cell membrane presents the antigen and the nanoparticle carriers the adjuvant. In this study, we used a genetically engineered cell membrane expressing SARS-CoV-2 RBD to coat biodegradable mesoporous silica nanoparticles (MSNs) that are encapsulated with cytosine-phosphate-guanine oligodeoxynucleotide (CpG) (Supplementary Fig. S1). Such RBD-displaying nanovaccine mimics the multivalent surface display of speci ﬁ c antigens by a viral article, with an effective antigen presentation process potentiated by the encapsulated adjuvant. After establishing that this nanovaccine can be produced in scalable manner using a ﬂ ash nanocomplexation (FNC) technique, we show that mice immunized with this nanovaccine developed high titers of


Synthesis of biomimetic coronavirus nanovaccine 7
The CpG loading was achieved with the MSN-to-CpG mass ratio of 5 to 1. The  CpG was obtained after stirring at 4 ℃ overnight. Cell membrane extraction experiment 9 was conducted as follows, after trypsinization, HEK293T cells were lysed and 10 homogenized by sonication, then the cell pellets were collected by ultracentrifugation, 11 finally suspended in DI water. The total membrane protein contents were quantified 12 using the BCA protein assay kits. 13 FNC and bulk sonication producing-nanoparticles were synthesized as follows: all 14 particles and cell membrane fragments were well dispersed in DI water, respectively. 15 In the FNC method, particle solutions and cell membrane fragments were introduced 16 into different inlets of the MIVM, respectively. The flow rate of 30 mL/min was applied 17 to prepare membrane-coated particles with a mass ratio of 1/1. The efflux was collected 18 for further use. For nanoparticles coated using the bulk sonication method, equal 19 volumes of cell membrane vesicles and particle cores were mixed, pipetted, and 20 sonicated. Nanoparticles of mixture formulation were prepared by mixing the CpG 21 loaded MSN and cell membrane at the equal mass after dispersion. 22 1

Characterization of biomimetic coronavirus nanovaccine 2
The size, polydispersity index (PDI) and zeta potential of naked MSN, cell membrane, 3 CpG loaded MSN, cell membrane-coated nanoparticles of FNC, bulk sonication and 4 mixture formulation were measured using a Malvern Zetasizer. To assess the stability 5 of CpG coated MSN, membrane-coated nanoparticles of FNC and bulk sonication 6 formulation, particles were stored in the FBS containing DMEM medium and PBS for 7 Dendritic cells (BMDCs) followed a previously published protocol. Healthy mice were 21 euthanized using carbon dioxide asphyxiation followed by cervical dislocation. Both femurs and tibias were dissected, cleaned in 75% ethanol, and cut on both ends. Bone 1 marrow was then flushed out of the bone with a 1 mL sterile syringe using warm PBS. 2 Cells were then pelleted at 700×g for 5 min, resuspended in a certain amount of red 3 blood cell lysis buffer to reduce the red blood cell. Cells were then pelleted at 700×g 4 for 5 min again, resuspended in BMDC growth media, consisting of the basal media 5 further supplemented with 20 ng/mL granulocyte/macrophage-colony stimulating 6 factor (GM-CSF) and 10 ng/mL interleukin-4 (IL-4), to a concentration of 1×10 6 7 cells/mL, and plated into Petri plates at 10×10 6 cells per 100 mm plate. The medium 8 was half-changed every two days. 9 10 6. Cytotoxicity assay 11 The cytotoxicity of naked MSN, cell membrane, CpG coated MSN, cell membrane-12 coated nanoparticles of bulk sonication, FNC and mixture formulation in the 13 RAW264.7 cells or BMDCs was assessed using a CCK8 assay. The cells in the proper 14 density were cultured in complete medium containing 5 or 20 μg/mL substances (CpG 15 formulation) for 24 h, and then incubated with CCK8 solution for 1 hour. The cell 16 activity was detected by reading the OD at 450 nm. 17 18

In vitro uptake and activity 19
For the cellular uptake study, BMDCs were collected on day 5 and then plated into 12-20 well plates. FITC-labeled CpG, DiD-labeled CM, MSN-CpG, CM-coated nanoparticles 21 of FNC, bulk and mixture formulation were added at an equivalent CpG concentration of 5 μg/mL. After 4 h incubation, the cells were washed and stained with DAPI. 15 min 1 later, cells were imaged by fluorescence. For flow cytometry, cells were collected, 2 washed twice in PBS, and resuspended in 300 µL PBS. The cell suspension was 3 analyzed using BD FACSCelesta flow cytometer. The activity of the delivered CpG 4 was examined using a BMDC maturation assay and cytokine release assay. BMDCs 5 were collected on day 5, and 5×10 5 BMDCs were plated into 6-well plates in BMDC 6 growth media. Cells were pulsed with materials for 12 h at 5 μg/mL CpG, then washed 7 twice with fresh media. After an additional 24 h of culture, cell supernatants were 8 collected and cytokine content was analyzed using TNF-α ELISA kits. The cells were 9 then collected, washed twice and stained with PE-conjugated CD11c, PerCP/Cy5.5-10 conjugated CD40, PE/Dazzele 594-conjugated CD80 and APC-conjugated CD86. Data 11 were collected using a BD FACSCelesta flow cytometer. RAW264.7 cells were plated 12 into 96-well suspension plates at 2×10 4 cells/well and pulsed with materials for 24 h at 13 5 μg/mL CpG, then cell supernatants were collected and cytokine content was analyzed 14 using TNF-α ELISA kits.  Table S2) for 30 min. Data were collected using BD FACSCelesta flow 7 cytometer and analyzed using FlowJo software. 8 9

Mouse vaccination experiments 10
Female 5-week-old BALB/c mice were randomly assigned to 5 cohorts of 11 cell membrane, mixture, CM-coated nanoparticles of FNC and bulk formulation, 12 vaccinated with about 500 ng of RBD subcutaneously and boosted on week 2, 13 respectively. Mice of control group were injected with normal saline of equal volume. 14 The weight of mice were monitored every week. On week 2, 4, 6, 8, 10 after prime 15 vaccination, mice were sacrificed and blood was collected in the coagulation-promoting 16 tubes. Plasma was separated by centrifugation and stored at -80 °C for further use. The 17 levels of TNF-α and IFN-γ in serum of vaccinated mice on week 4 were detected by 18 ELISA kits. 19 To assess RBD-specific T cells, mice were sacrificed on the week 4 and 20 splenocytes were collected. Single-cell suspensions of splenocytes were prepared by 21 gently grinding. Splenocytes were stained for lymphocyte, macrophages, DCs, Tc cells, Th cells and the subtypes (panel shown as Table S1) for 30 min. Then, stained cells 1 were incubated with 10 μg/mL DiD-labeled MSN-CpG@CM (FNC) binding to the 2 RBD-specific T cells. Data were collected using BD FACSCelesta flow cytometer and 3 analyzed using FlowJo software. 4 To evaluate the safety of vaccines, the main tissues including livers, kidneys, 5 spleens, lungs and hearts from the vaccinated mice on week 10 were collected, fixed in 6 4% formalin and sectioned for hematoxylin and eosin (H&E) staining. The biochemical 7 parameters including alanine aminotransferase (ALT), aspartate aminotransferase 8 (AST), Urea, creatinine (CREA) and total protein (TP) were assayed. 9 10

RBD-specific IgG antibody detection and neutralization test of pseudovirus 11
Antibody titer detection adopted indirect ELISA method. In short, RBD protein (1 12 μg/ml) was coated on the well plate and stored overnight at 4 ℃. And then plate was 13 washed three times. The serum of vaccinated mice on week 2, 4, 6, 8, 10 were serial 14 diluted and added into the pre-coated well plate for incubation (37 ℃, 1 h). And then 15 the PBS was added to wash the well three times. Next, the enzyme labeled antibody 16 was added and incubated for one hour. And then the PBS was added to wash the well 17 three times. TMB solution was added to incubate for near 15 minutes in the dark. 18 Finally, the stop solution was added, and the absorbance at 450 nm was measured with 19 a microplate reader. The absorbance data was simulated in nonlinear fitting by Origin 20 2021b software. The positive data was confirmed 2.1 times larger than the negative data.
In the pseudovirus neutralization test, 20 μL of serum of vaccinated mice on week 1 4 was first added and incubated with 20 μL of SARS-CoV-2 Spike pseudovirus with 2 GFP expressing the gene for 30 min and then the mixture was added to HEK293T-3 ACE2 cells to 200 μL total culture medium. After 24 h of co-cultivation, the 4 intracellular fluorescence content was measured by fluorescence to verify the protective 5 effect of the serum RBD-specific antibody against pseudovirus. 6 7

Statistical analysis 8
Statistical analysis was performed using GraphPad Prism 9 (GraphPad). Data were 9 analyzed using one-way ANOVA with Tukey's post-hoc correction for multiple 10 hypothesis testing unless otherwise stated. All flow cytometry data were analyzed using 11